[The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction]

Objective: To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs).

Methods: The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall method in vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×10 ⁵ cells/mL); group B, BMSCs (1×10 ⁵ cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×10 ⁵ cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×10 ⁵ cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×10 ⁵ cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot.

Results: The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance ( A) values had significant differences between groups ( P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E ( P<0.05), but there was no significant difference among groups A, D, and E ( P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A ( P<0.05); but there was no significant difference between groups D and E ( P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups ( P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups ( P<0.05).

Conclusions: GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.